The following description is provided to assist the understanding of the reader. None of the information provided or references cited is admitted to be prior art in the present invention.
Poymerase Chain Reaction (PCR) is likely the most widely used method in modern molecular biology and biotechnology, and is rapidly being applied to genetic testing, diagnostics, forensics and biodefense. Kolmodin, L. A. et al., Nucleic Acid Protocols Handbook, 569-580 (Rapley, R. ed., Humana Press 2000); Budowle, B., et al., 301 Science, 1852-1853 (2003); Sato, Y. et al., 5 (Suppl. 1) Legal Medicine, S191-S193 (2003); Saldanha. J., et al., 43 J. Medical Virol., 72-76 (1994); Dahiya. R., et al., 44 Biochemistry and Molecular Biology International, 407-415 (1998); and Elnifro, E. M., et al., 13 Clin. Microbiol. Rev., 559-570 (2000). PCR is described in U.S. Pat. Nos. 4,683,195 and 4,683,202. In each cycle of the PCR amplification process there are typically several steps. The double-stranded DNA target sequence is first thermally denatured at elevated temperatures (˜95° C.). The first occurrence of denaturation is oligonucleotide primer to each strand at lower temperatures (˜60° C.). These forward and reverse oriented oligonucleotide primers are then each extended from their 3′-termini at an elevated temperature (˜70° C.) by a thermally stable, magnesium ion-dependent, DNA polymerase which incorporates 2′-deoxyribonucleoside 5′-triphosphates (dNTPs) and generates pyrophosphate (PPi).
The utility of PCR is driven by its ability to rapidly provide target amplifications of ˜106-fold as well as high specificity, which depends in part on the specificity of oligonucleotide primer hybridization. Oligonucleotide primer sequences and length are therefore designed to hybridize to only the intended target sequence, at the temperatures used for annealing. However, PCR amplification reactions are typically prepared over a period of minutes or hours at ambient room temperatures which are well below the temperature range needed to ensure specificity of oligonucleotide primer hybridization. Under such low stringency sample preparation conditions and following an initial pre-PCR denaturation step, oligonucleotide primers may bind non-specifically to other sequences and potentially initiate synthesis of undesired extension products, which can be amplified along with the target sequence. Amplification of non-specific target sequences having partial complementarity to the primers, so called “mis-priming,” can compete with amplification of desired target sequences, and can significantly decrease efficiency of amplification of the desired sequence, especially for low-copy number targets (Chou, Q., et al., 20 Nucleic Acids Res. 1717-1723 (1992)).
Formation of “primer dimers” is another problematic form of non-specific hybridization, which, according to Chou, Q., et al., results from amplification of two oligonucleotide primers extended across one another's sequence without significant intervening sequence. These investigations further noted that primer dimers may undergo amplified oligomerization during PCR to create a complex mixture of oligonucleotide primer artifacts, the quantity and quality of which often varies inversely with the yield of specific PCR product in low copy number amplifications.
While the aforementioned problems due to mis-priming and primer dimer formation can be encountered in all applications of PCR, these issues can be particularly challenging for high-sensitivity analytical PCR schemes, such as those used for detection of blood-borne infectious agents (Saldanha, J., et al.; Elnifro, E. M., et al.), biohazardous microbes (Budowle, B., et al.), defective or cancerous genes (Dahiya, R., et al.), and forensics (Budowle, B., et al.; Y. Sato, et al.). In addition, there is a much greater chance for formation of spurious amplification products in multiplex PCR. Markoulatos, P., et al., 16 J. of Clin. Laboratory Analysis, 47-51 (2002). In reverse transcriptase PCR (RT-PCR), the most sensitive means for detection of a target RNA sequence is to use a gene-specific oligonucleotide primer in the RT step. Zhang, J., et al., 337 Biochem. J., 231-241 (1999); Lekanne Deprez, R. H., et al., 307 Analytical Biochem., 63-69 (2002); Bustin, S, A., et al., 15 J. of Biomolecular Techniques, 155-166 (2004). In view of the importance of these high-sensitivity applications requiring high specificity to avoid serious, adverse consequences of “false negatives” and “false positives,” it is critical to have reagents and protocols which provide assays that are functionally free of artifacts due to mis-priming and primer dimer formation.
A number of general strategies have been investigated for reducing non-specific amplification based on the so-called “hot start” process which aims at impairing undesired amplification due to mis-priming and oligonucleotide primer dimer formation under low-stringency conditions e.g., at room temperature during sample preparation and following an initial pre-PCR denaturation step. Amplification subsequently begins when the reaction mixture reaches high-stringency, i.e., “hot” temperatures to “start” polymerase-mediated extension of oligonucleotide primers hybridized only to target sequences. Thus temperature triggers enzymatic extension of oligonucleotide primers only at elevated temperatures when the stringency of primer/target hybridization conditions is optimal for specificity.
These general strategies for “hot start” include the use of (1) temperature-sensitive materials, such as waxes as barriers or sequestrants to control mixing of the reagents (Chou, Q., et al.; Tanzer, L. R., et al., 273 Analytical Biochem., 307-310 (1999)); (2) oligonucleotide aptamers (Dang, C., et al., 264 J. Mol. Biol., 268-278 (1996)) or antibodies (Eastlund, E., et al., 2 LifeScience Quarterly, 2-5 (2001); Mizuguchi, H., et al., 126 J. Biochem. (Tokyo), 762-768 (1999)) that inhibit the function of DNA polymerases; (3) use of a second thermostable enzyme, such as pyrophosphatase (Clark, D. R., et al., International Patent Application No. WO 2002088387) to remove suppression by added pyrophosphate (PPi); (4) chemically modified polymerases with hydrolytically reversible reagents, such as citraconic acid-modified lysine (Birch, D. E., et al., U.S. Pat. No. 5,773,258) in AmpliTaq Gold (Moretti, T., et al., 25 BioTechniques, 716-722 (1998); Saldanha, J., et al.); (5) oligonucleotide primer sequence constructs that disfavor low-temperature mis-priming, such as competitor sequences (Puskas, L. G., et al., 5 Genome Research, 309-311 (1995)) or “touch-up and loop-incorporated oligonucleotide primers” (TULIPS-PCR) (Ailenberg, M. et al., 29(5) BioTechniques, 1018-1023 (2000)); and (6) chemically modified primer containing phosphotriester internucleotide linkage(s) near the 3′-end of the primer (i.e., phosphotriester primers) (Zon, G., et al., U.S. Patent Appl. No. 20070281308 (2007)).